Going Full TeRM: The Seminal Role of Tissue-Resident Macrophages in Organ Remodeling during Pregnancy and Lactation.

During pregnancy and lactation, the uterus and mammary glands undergo remarkable structural changes to perform their critical reproductive functions before reverting to their original dormant state upon childbirth and weaning, respectively. Underlying this incredible plasticity are complex remodeling processes that rely on coordinated decisions at both the cellular and tissue-subunit levels. With their exceptional versatility, tissue-resident macrophages play a variety of supporting roles in these organs during each stage of development, ranging from maintaining immune homeostasis to facilitating tissue remodeling, although much remains to be discovered about the identity and regulation of individual macrophage subsets. In this study, we review the increasingly appreciated contributions of these immune cells to the reproductive process and speculate on future lines of inquiry. Deepening our understanding of their interactions with the parenchymal or stromal populations in their respective niches may reveal new strategies to ameliorate complications in pregnancy and breastfeeding, thereby improving maternal health and well-being.

Mammary duct luminal epithelium controls adipocyte thermogenic programme.

Sympathetic activation during cold exposure increases adipocyte thermogenesis via the expression of mitochondrial protein uncoupling protein 1 (UCP1)1. The propensity of adipocytes to express UCP1 is under a critical influence of the adipose microenvironment and varies between sexes and among various fat depots2-7. Here we report that mammary gland ductal epithelial cells in the adipose niche regulate cold-induced adipocyte UCP1 expression in female mouse subcutaneous white adipose tissue (scWAT). Single-cell RNA sequencing shows that glandular luminal epithelium subtypes express transcripts that encode secretory factors controlling adipocyte UCP1 expression under cold conditions. We term these luminal epithelium secretory factors 'mammokines'. Using 3D visualization of whole-tissue immunofluorescence, we reveal sympathetic nerve-ductal contact points. We show that mammary ducts activated by sympathetic nerves limit adipocyte UCP1 expression via the mammokine lipocalin 2. In vivo and ex vivo ablation of mammary duct epithelium enhance the cold-induced adipocyte thermogenic gene programme in scWAT. Since the mammary duct network extends throughout most of the scWAT in female mice, females show markedly less scWAT UCP1 expression, fat oxidation, energy expenditure and subcutaneous fat mass loss compared with male mice, implicating sex-specific roles of mammokines in adipose thermogenesis. These results reveal a role of sympathetic nerve-activated glandular epithelium in adipocyte UCP1 expression and suggest that mammary duct luminal epithelium has an important role in controlling glandular adiposity.

Graduate Student Literature Review: The challenge of drying-off high-yielding dairy cows.

The cessation of lactation (i.e., dry-off) in dairy cattle is an area of research that has received much focus in recent years. The dry period is necessary to optimize tissue remodeling of the mammary gland, but represents a stressful event, incorporating several changes in daily routine, diet, and metabolism. Moreover, the high milk yields achieved by modern cows in late gestation exacerbate the need for relevant manipulations in the days around dry-off, as excessive accumulation of milk might jeopardize the success of the dry period, with potential negative effects on future lactation. Production levels over 15 kg/d are an additional risk factor for udder health, delay mammary involution, and worsen metabolic stress and inflammatory responses. Furthermore, the pressure to reduce antibiotic usage in farm animals has resulted in increased attention on the dry period, given that historically most dairy cattle were provided prophylactic intramammary antibiotic treatment at dry-off as a means to reduce the risk of intramammary infections in the subsequent lactation. Several strategies have been proposed over the years to cope with these challenges, aiming to gradually reduce milk yield before dry-off, promoting at the same time the start of mammary involution. Among them, the most common are based on feed or nutrient restriction, a decrease in milking frequency, or administration of prolactin inhibitors. These practices have different capacities to reduce milk yield through different mechanisms and entail several implications for udder health, animal welfare, behavior, endocrine status, metabolism, and inflammatory conditions. The present review aims to provide a comprehensive overview of the dry-off phase in high-yielding cows and of the impact of high milk production at dry-off, and to describe possible strategies that might be implemented by farmers and veterinarians to optimize this critical phase in an integrated way.

A randomized controlled trial to study the effects of an automated premilking stimulation system on milking performance, teat tissue condition, and udder health in Holstein dairy cows.

The objectives were to examine the effect of an automated premilking stimulation (APS) by means of a high pulsation frequency (300 cycles/min) without a reduction of the vacuum in the pulsation chamber or claw piece on (1) milking performance, (2) teat tissue condition, and (3) udder health in dairy cows. In a randomized controlled field study, Holstein cows (n = 427) from 1 commercial dairy farm with a milking schedule of 3 times per day were assigned to treatment and control groups over a 90-d period. Treatments consisted of a maximum of 80 s (APS80) or 99 s (APS99) of mechanical stimulation at a pulsation rate of 300 pulses per minute and a ratio of 25:75 (no reduction of the pulsation chamber or milking vacuum). Cows in the control group (CON) received traditional premilking stimulation by means of manual forestripping for 8 s. Milking characteristics were documented with on-farm milk meters. Short- and long-term changes in teat tissue condition induced by machine milking were assessed visually on a weekly basis. Composite milk samples were analyzed once per month to determine somatic cell count. Generalized linear mixed models were used to study the effect of the treatment on the outcome variables. We observed no meaningful differences in milk yield or milking unit-on time. Least squares means and their 95% confidence intervals (95% CI) for cows in the APS80, APS99, and CON groups were 13.5 (13.1-14.0), 13.2 (12.8-13.7), and 13.2 (12.8-13.7) kg for milk yield and 222 (213-231), 219 (210-228), and 223 (214-232) s for milking unit-on time, respectively. The effect of treatment on bimodality was modified by milk yield such that the odds of bimodality increased in the treatment groups with increasing milk yield. Compared with cows in the CON group, the odds ratios (95% CI) of bimodality were 1.08 (0.62-1.89) in the APS80 group and 0.89 (0.55-1.42) in the APS99 group at a milk yield of 11 kg and 2.0 (1.24-3.22) in the APS80 group and 2.08 (1.29-3.35) in the APS99 group at a milk yield of 16 kg. We observed differences in short- and long-term changes in teat tissue condition between the treatment and control groups. Compared with cows in the CON group, the odds (95% CI) of short-term changes were 1.87 (1.35-2.58) for the APS80 group and 1.49 (1.08-2.07) for the APS99 group, and the odds of long-term changes were 1.52 (1.24-1.85) for cows in the APS80 group and 1.59 (1.31-1.94) for cows in the APS99 group. The least squares means (95% CI) for somatic cell counts (log10-transformed) were 4.74 (4.68-4.81) for the APS80 group, 4.77 (4.71-4.83) for the APS99 group, and 4.79 (4.73-4.86) for the CON group. We conclude that the APS system tested here had no negative effects on milk yield or milking unit-on time. However, differences in bimodality and teat tissue condition suggest that the APS system did not provide sufficient stimulation to facilitate a gentle milk harvest and adversely affects teat tissue condition.

Taurine alleviates heat stress-induced mammary inflammation and impairment of mammary epithelial integrity via the ERK1/2-MLCK signaling pathway.

Heat stress leads to milk production losses and mammary gland inflammation, which may be associated with mammary epithelium damage. Taurine is one of the most abundant free amino acids in mammals which has anti-inflammatory properties. This study aimed to explore the effect of taurine pretreatment on heat stress-induced mammary epithelial integrity disruption and inflammatory damage. In our first experiment on dairy cows our results showed that compared with animals under autumn thermoneutral condition (THI = 62.99 ± 0.71), summer heat stress (THI = 78.01 ± 0.39) significantly reduced milk yield and disrupted mammary epithelial integrity as revealed by increased concentrations of serotonin and lactose in plasma, and increased levels of SA and Na+/K+ in milk. In our second study, 36 lactating mice were randomly divided into three groups (n = 12) for a 9d experiment using a climate chamber to establish a heat stress model. Our findings suggest taurine pretreatment could attenuate heat stress-induced mammary histopathological impairment, inflammation response, and enhance mammary epithelium integrity, which was mainly achieved by promoting the secretion of ZO-1, Occludin, and Claudin-3 through inhibiting activation of the ERK1/2-MLCK signaling pathway in the mammary gland. Overall, our findings indicated that heat stress induced mammary epithelium dysfunction in dairy cows, and emphasized the protective effect of taurine on mammary health under heat stress conditions using a mouse model, which may be achieved by alleviating the mammary epithelium integrity damage and inflammation response.

Is the temperament of crossbred dairy cows related to milk cortisol and oxytocin concentrations, milk yield, and quality?

Reactive dairy cows are more susceptible to stress, and this may result in negative effects on milk yield and quality. The aims of this study were to investigate the relationships between temperament traits and concentration of milk cortisol and oxytocin, milk yield, milkability, and milk quality in Holstein-Gyr cows. Temperament traits were assessed in 76 Holstein-Gyr cows in the milking parlor (by scoring milking reactivity and recording the numbers of steps and kicks during pre-milking udder preparation and when fitting the milking cluster) and during handling in the corral (by measuring the time to enter in the squeeze chute, ET and flight speed, FS). Milk samples were collected for milk quality (% fat, % protein, % lactose, and somatic cell count, SCC), and milk cortisol and oxytocin. Milk yield, milking time, and average flow were also measured. The calmer cows during milking management (class 'low') produced milk with higher protein (p = 0.028) content and tendencies for lower fat (p = 0.056) and higher lactose (p = 0.055) contents. Regarding the hormones, the most reactive cows (class 'high') in the milking and handling corral produced milk with higher concentrations of cortisol (p<0.001) and oxytocin (p = 0.023). In addition, the temperament of the animals affected some of the productive measures evaluated. Cows with reactive temperament had lower milk flow and longer milking time than the intermediate ones and had higher fat and a tendency for lower protein percentage in milk compared to cows with intermediate temperaments. Calm and intermediate cows in the handling corral produced more milk and presented better milkability parameters, such as a shorter milking time and greater average milk flow. Our results suggest that the cows' behavioral reactivity can be related to the intensity of their response to stress during handling.

Relationship between blood calcium level and post-milking teat canal closure in dairy cows.

The teat canal-one of the primary defense mechanisms of the udder-ensures the milk flow during milking in bovines and prevents pathogens from entering the udder by forming a barrier through the elastic muscle and keratin layers tightly closing the surrounding area. The current study investigated the effects of blood calcium status on teat closure in cows after milking. The study covered 200 healthy teats, of which 100 were from normocalcemic (NC) cows and 100 were from subclinical hypocalcemic (SCH) cows. Teat canal length (TCL) and width (TCW) were measured with ultrasonography at 0-min pre-milking and 15- and 30-min post-milking. Cylindrically shaped teat canal volume (TCV) was calculated by deriving from TCL and TCW. Time-dependent changes in teat canal closure and their relationships with blood calcium levels were analyzed. The results showed that the calcium level did not affect TCL, TCW, and TCV (P > 0.05) during the 15-min post-milking period. However, TCL (P < 0.001), TCW (P < 0.05), and TCV (P < 0.001) were lower in NC cows than in SCH cows at 30-min post-milking. At 15-min post-milking, no correlation existed between the teat canal closure (ΔTCL, ΔTCW, and ΔTCV) and the blood calcium level, while significant correlations were available between the teat canal closure and the blood calcium level {ΔTCL (r: - 0.288, P < 0.001), ΔTCW (r: - 0.260, P < 0.001), ΔTCV (r: - 0.150, P < 0.05)} at 30-min post-milking. The current study concluded that the blood calcium status significantly impacts the teat canal closure in bovines, and calcium status should be meticulously monitored with the mastitis control program to apply necessary strategic steps.

The interplay of continuous milk ejection and milking system with and without prestimulation at different vacuum settings.

Efficient machine milking requires an optimal interaction of alveolar milk ejection in the udder and milk removal by the milking machine. The aim of the present study was to test whether the equilibrium between continuous milk ejection and milk removal can also be maintained at very fast milking through a particularly high vacuum. Eight Holstein dairy cows were milked at 42, 52, or 60 kPa, with (PS) or without (nPS) prestimulation. Each of the 6 treatments was conducted at 2 afternoon milkings in each animal. The prestimulation lasted 40 s and consisted of forestripping and teat cleaning. The cluster attachment followed after a 20-s latency period. Throughout each milking, B-mode ultrasound videos of the gland cistern of 1 front quarter as well as milk flow and claw vacuum curves were recorded. Total milk yield was neither affected by nPS or PS nor by the vacuum level. Milk removed within the first minute and the first 2 min of milking and average milk flow were higher, and the duration of incline and time until peak milk flow were shorter at PS than at nPS milkings at all vacuum levels. Machine-on time was shorter at PS than at nPS milkings, although only at 42 and 52 kPa vacuum, obviously caused by the high percentage of bimodalities occurring in nPS milkings (17% bimodalities in PS vs. 92% bimodalities in nPS milkings). The frequency of bimodalities was higher at high than at low vacuum both in PS and nPS milkings. Peak flow rate and average milk flow were both higher at higher vacuum levels. The duration of milk flow plateau was shorter at 60 kPa than at 42 kPa milkings. At the highest vacuum (60 kPa), the shorter plateau phase indicated a declining milk ejection rate toward the end of the plateau phase, and milk ejection could no longer keep up with the fast milk removal; hence, a higher milking efficiency at a higher vacuum level could only be achieved as long as the gland cistern remained sufficiently filled by the continuous milk ejection. The ultrasound imaging confirmed this finding as the duration of cisternal area plateau in the recorded front quarter was shorter at high than at low vacuum. Thus, the highest vacuum of 60 kPa did not cause a shorter machine-on time than 52 kPa. In conclusion, milking at a very high vacuum can increase milking efficiency compared with a low vacuum. However, a vacuum reduction at the start and toward the end of milking is required to prevent overmilking if milking is performed at a very high vacuum.

Bimodal milk flow and overmilking in dairy cattle: risk factors and consequences.

To maximise the return on capital invested in the milking parlour, the largest number of cows should be milked gently and completely in the shortest possible time. Bimodal milk flow and overmilking negatively influence the efficiency of the milk removal process and teat health. This observational study had the objective of investigating the prevalence of bimodal milk flow and overmilking, determining which individual and farm-related variables are associated with these occurrences, and determining the association of overmilking and bimodal milk flow with milk yield and with short- and long-term teat changes. Twenty-one farms were visited once during the study period, wherein the milking routine was timed, the teat condition was assessed, and dynamic evaluation of the milking vacuum was performed. A total of 606 vacuum graphic records were obtained, with an average of 29 ± 3 records per farm, in order to indirectly evaluate the milk flow and thus determine the occurrence of bimodal milking and overmilking time. The average percentage of bimodality per farm was 41.7%. The median overmilking time was 59 seconds, and on average, 78.3% of the cows in a herd were overmilked longer than 30 seconds. An association was found at cow level between the occurrence of bimodal milk flow and days in milk, the total stimulation time, parity, and the preparation lag time. The increase in the mean total stimulation time and the number of passes during preparation were associated with a decrease in the proportion of bimodality in the herd. Parity, reattachment of the milking unit and milking in manual mode were associated with an increase in overmilking time of an individual cow. The presence of a clogged air bleed hole in the claw and the reduction of the cluster removal milk flow threshold were associated with an increase in the herd's median overmilking time. The average milk flow decreased with the increase in overmilking time and with the occurrence of bimodal milk flow. An association was also found between the occurrence of bimodal milk flow and decreased milk yield. A mean of 78.4% of cows per farm had short-term teat changes in at least one teat, and 33.6% of evaluated cows per farm displayed at least one teat with hyperkeratosis. These results emphasise the association of bimodality and overmilking on milking efficiency and reinforce the importance of the milkers' actions and the functioning of the milking parlour for its prevention.

Effect of mechanical premilking stimulation on milking duration in late lactation.

This study documents the effect of mechanical prestimulation on the milking duration of pasture-based cows in late lactation to better harness increased capacity of automation in the milk harvesting process. Premilking stimulation, provided via manual or mechanical means, has been shown to promote the milk letdown reflex and assist in achieving quick, comfortable, and complete milk removal from the udder. The literature is lacking knowledge on the effect of mechanical premilking stimulation on milking duration, especially in late lactation and in pasture-based systems, and many pasture-based farms do not practice a full premilking routine because of a lack of labor availability. The current study addresses this gap in knowledge. In this study, we tested 2 treatments: (1) the No Stim treatment used normal farm milking settings with no premilking preparation and (2) the Stim treatment used 60 s of mechanical premilking stimulation, with a rate of 120 cycles per minute and a pulsator ratio of 30:70 on cluster attachment. Once the 60 s of stimulation had elapsed, normal milking settings resumed for the remainder of the milking. Sixty cows were enrolled in the study, which ran for 20 d. The effect of treatment on a.m. milking duration was significant, a.m. milking duration for Stim was 12 s shorter than that of No Stim. The effect of treatment on p.m. milk duration was not significant. Treatment had no effect on a.m./p.m. milk yields, average milk flowrates or peak milk flowrates. Significant differences emerged between treatments on a.m. and p.m. dead time (time from cluster attachment to reach a milk flowrate of 0.2 kg/min). The a.m. and p.m. dead times were 6 s shorter for Stim compared with No Stim. The time taken to achieve peak milk flowrate (time to peak) at morning milking was 7 s shorter for Stim compared with No Stim, and treatment yielded no significant effects on time to peak at p.m. milkings. Treatment also had no significant effect on log10 somatic cell count. Although the percentage of congested teat-ends and teat-barrels was numerically lower for Stim versus No Stim, no statistical differences were detected across these measures. Based on the results of the study, we found merit in applying 60 s of mechanical pre-stimulation at a.m. milking from a milking duration perspective. However, the strategy was not as successful for the p.m. milking. Analysis of the milk flowrate profiles recorded during the study suggest potential utility in employing different machine settings for various milkings based on anticipated yield and level of udder fill.

Adult Stem Cell Research in Light of the Bovine Mammary Gland Regenerative Medicine.

The bovine mammary gland has vital importance in the dairy sector, as it is considered a source of basic dairy product, milk. Mammary gland affections are widespread, which affect the dairy industry economically and pose a potential public health hazard. Current therapeutic options are ineffective in controlling the infection and regenerating the gland effectively. Antimicrobials commonly used against mastitis make their way into the milk . In order to find a solution to these problems, advanced therapeutic options, like the one for stem cells, are considered. Mammary gland stem cells (MaSCs) are considered to maintain tissue homeostasis. The characterization of these cells and their derived lineages (progenitor cells and mammary epithelial cells) may potentially provide the physiological status or production potential of the gland. However, current isolation methods are cumbersome and fall short to isolate a pure line of cattle MaSCs from progenitors or other differentiated epithelial cells. An alternative to the therapeutic application of MaSCs is the mesenchymal stem cell (MSC). These cells can potentially control microbial infection, show anti-inflammatory and other pro-healing effects, and furthermore enhance mammary epithelial cell secretory potential to ensure tissue regeneration. The current review focuses on MaSCs and MSCs properties in light of the bovine mammary gland regeneration.

Symposium review: Environmental effects on mammary immunity and health.

Environmental effects on pathogen abundance and access are precursors to mastitis. Indeed, high heat and humidity, and unsanitary housing and equipment, are associated with greater pathogen load and exposure. Although less is known about effects of environment on a cow's ability to resist infection, several indicators suggest that it can affect pathogen responses. Mastitis incidence and bulk tank somatic cell count vary with season, typically peaking in summer. Recent controlled studies have revealed that heat stress exposure results in changes in the microbiome of the cow and her environment, which may relate to negative effects on milk quality and cow health. Alternatively, specific pathogen loads may vary based on housing dynamics rather than associations with physical environment. Indeed, housing-related stressors, such as overcrowding and social group challenge, influence secretion of glucocorticoids, thus affecting pathogen resistance in the cow. Two key seasonal variables are photoperiod and temperature, specifically the heat stress consequent to elevated temperature and humidity. Shifts in light duration regulate immune function in other species, but apparently have limited effect on udder health of lactating cows. In contrast, in dry cows, short days increase peripheral blood mononuclear cell number and are associated with lower somatic cell count in the next lactation, compared with long days. With heat stress, elevated body temperature directly affects expression of immune-related genes in mammary tissue. Responses depend on duration of exposure and feature acute upregulation of immune-signaling pathways, followed by enrichment of other immune-related pathways after prolonged exposure. Most responses are transient and recover within 1 wk. Functionally, heat stress impairs some aspects of acquired immunity in dry cows, including antigen responses and lymphocyte proliferation, but apparently not innate immune function. However, heat stress in late gestation reduces neutrophil phagocytosis and killing in vitro, and neutrophils in circulation are reduced in vivo as are responses to pathogen challenge in the subsequent lactation. A holistic understanding of the complex interplay of environment, pathogens, and host is needed to inform advances in this area.

Risk factors for delayed milk ejection in Holstein dairy cows milked 3 times per day.

Delayed milk ejection, manifested most often as bimodal milk flow, occurs when the cisternal milk fraction is removed before the alveolar milk reaches the gland cistern. It is thought to be a consequence of not meeting cows' physiological needs, due to insufficient premilking teat stimulation, inadequate timing of milking unit attachment, or both. It has been associated with decreased milking efficiency, reduced milk yield, and impaired teat and udder health. Traditionally, portable electronic milk meters have been used to assess the presence of delayed milk ejection in dairy cows. By contrast, incremental milk flow rates from on-farm milk meters and their suitability as a measure to assess delayed milk ejection have not been studied by rigorous methods. The objectives were (1) to describe a protocol for identification of cows with chronically delayed milk ejection (CDME) and (2) to investigate risk factors for CDME using incremental milk flow rates obtained from automated on-farm milk meters. In a retrospective case control study, milk flow data from a 4,300-cow dairy with a thrice-daily milking schedule were obtained over a 1-wk period. Incremental milk flow rates (0-15 s, 15-30 s, 30-60 s, and 60-120 s) were used to identify cows with delayed milk ejection. Cases of CDME were defined as presence of delayed milk ejection at all 21 milking observations. Cows that had no delayed milk ejection at any of the same 21 milking observations were included as controls. A total of 171 cases and 393 controls were included in the study based on these criteria. A logistic regression model was used to evaluate associations of the following risk factors with CDME: parity (1, 2, ≥3), stage of lactation (<100, 101-200, >200 DIM), presence of a nonlactating quarter, milk somatic cell count, average daily milk production, and health and management events. Parity and CDME were associated such that compared with cows in their third or greater lactation, the odds (95% confidence intervals, 95% CI) of CDME were 1.27 (0.71-2.25) for cows in their first and 4.77 (2.47-9.22) for animals in their second lactation. The odds of CDME increased with increasing stage of lactation, with an odds ratio of 0.20 (0.11-0.36) for early and 0.28 (0.15-0.52) for mid-lactation animals, respectively, compared with late lactation cows. A 1-kg increase in average daily milk production was associated with decreased odds of CDME [odds ratio (95% CI): 0.89 (0.87-0.92)]. A lameness event during the study period increased the odds of CDME [odds ratio (95% CI): 8.04 (1.20-53.83)], as did a vaccination event 1 wk before the study period [odds ratio (95% CI): 4.07 (0.99-16.71)]. This study confirmed associations between CDME and previously reported risk factors and identified several previously less rigorously investigated health and management events that could be associated with CDME. Incremental milk flow rates from individual cows serve as an automated tool to evaluate milk flow dynamics. This information could be used to improve individual premilking udder preparation to meet the animal's physiological requirements, improve teat and udder health, and enhance parlor efficiency.

Carry-over effects of dry period heat stress on the mammary gland proteome and phosphoproteome in the subsequent lactation of dairy cows.

Exposure to heat stress during a cow's dry period disrupts mammary gland remodeling, impairing mammary function and milk production during the subsequent lactation. Yet, proteomic changes in the mammary gland underlying these effects are not yet known. We investigated alterations in the mammary proteome and phosphoproteome during lactation as a result of dry period heat stress using an isobaric tag for relative and absolute quantitation (iTRAQ)-based approach. Cows were cooled (CL; n = 12) with fans and water soakers in a free stall setting or were heat stressed through lack of access to cooling devices (HT; n = 12) during the entire dry period (approximately 46 days). All cows were cooled postpartum. Mammary biopsies were harvested from a subset of cows (n = 4 per treatment) at 14, 42, and 84 days in milk. Overall, 251 proteins and 224 phosphorylated proteins were differentially abundant in the lactating mammary gland of HT compared to CL cows. Top functions of differentially abundant proteins and phosphoproteins affected were related to immune function and inflammation, amino acid metabolism, reactive oxygen species production and metabolism, tissue remodeling, and cell stress response. Patterns of protein expression and phosphorylation are indicative of increased oxidative stress, mammary gland restructuring, and immune dysregulation due to prior exposure to dry period heat stress. This study provides insights into the molecular underpinnings of disrupted mammary function and health during lactation arising from prior exposure to dry period heat stress, which might have led to lower milk yields.

Orthotopic Transplantation of Mouse Mammary Epithelial Cells.

The orthotopic transplantation assay has provided important insights into mammary development, stem cell function, and tumorigenesis. Technically, it consists in grafting mammary tissue fragments, organoids, mammospheres, or isolated cells into the fat pads of prepubertal mice from which the endogenous epithelium has been surgically removed, thereby allowing growth and differentiation of mammary epithelial cells in their physiological environment. Here, we describe how is conducted transplantation of epithelial fragments and cells isolated from mouse mammary glands, report the various approaches currently used to evaluate the regeneration and self-renewal properties of mammary stem cells, and highlight the strengths and limitations of this in vivo grafting assay.

A physiologically based pharmacokinetic (PBPK) model exploring the blood-milk barrier in lactating species - A case study with oxytetracycline administered to dairy cows and goats.

Antibiotic excretion into milk depends on several factors such as the compound's physicochemical properties, the animal physiology, and the milk composition. The objective of this study was to develop a physiologically based pharmacokinetic (PBPK) model describing the passage of drugs into the milk of lactating species. The udder is described as a permeability limited compartment, divided into vascular, extracellular water (EW), intracellular water (IW) and milk, which was stored in alveolar and cistern compartments. The pH and ionization in each compartment and the binding to IW components and to milk fat, casein, whey protein, calcium, and magnesium were considered. Bidirectional passive diffusion across the blood-milk barrier was implemented, based on in vitro permeability studies. The model application used to predict the distribution of oxytetracycline in cow and goat milk, after different doses and routes of administration, was successful. By integrating inter-individual variability and uncertainty, the model also allowed a suitable estimation of the withdrawal periods. Further work is in progress to evaluate the predictive ability of the PBPK model for compounds with different physico-chemical properties that are potentially actively transported in order to extrapolate the excretion of xenobiotics in milk of various animal species including humans.

Alveolar progenitor differentiation and lactation depends on paracrine inhibition of notch via ROBO1/CTNNB1/JAG1.

In the mammary gland, how alveolar progenitor cells are recruited to fuel tissue growth with each estrus cycle and pregnancy remains poorly understood. Here, we identify a regulatory pathway that controls alveolar progenitor differentiation and lactation by governing Notch activation in mouse. Loss of Robo1 in the mammary gland epithelium activates Notch signaling, which expands the alveolar progenitor cell population at the expense of alveolar differentiation, resulting in compromised lactation. ROBO1 is expressed in both luminal and basal cells, but loss of Robo1 in basal cells results in the luminal differentiation defect. In the basal compartment, ROBO1 inhibits the expression of Notch ligand Jag1 by regulating ß-catenin (CTNNB1), which binds the Jag1 promoter. Together, our studies reveal how ROBO1/CTTNB1/JAG1 signaling in the basal compartment exerts paracrine control of Notch signaling in the luminal compartment to regulate alveolar differentiation during pregnancy.

Generation and functional characterization of murine mammary organoids.

3D cultures of mammary epithelial cells purified from murine models provide a unique resource to study genetically defined breast cancer and response to targeted therapies. Here, we describe step-by-step experimental procedures for the successful establishment of murine mammary organoid lines isolated from mammary glands or mammary tumors driven by mutations in components of the PI3K pathway. These detailed protocols also include procedures to perform assays that can be adopted to screen response to drug treatments and to inform better therapies. For details on potential applications and use of this protocol, please refer to Yip et al. (2020).

3D in vitro culture system to study collective migration in mammary organoid epithelium.

We recently established an in vitro culture system in which mammary gland organoid undergoes directional migration in response to an FGF10 concentration gradient. Here, we describe a step-by-step protocol for preparing organoids, the setup of the 3D culture system, and the image acquisition approach. The technical difficulties in conducting the 3D migration assay are choosing epithelial organoids of appropriate sizes and manually paring organoids and beads pre-soaked in FGF10 within a desirable distance (∼100 µm). For complete details on the use and execution of this protocol, please refer to Lu et al. (2020).

Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis.

The role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to ChIP-seq peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). RT-qPCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.